A continuous microplate assay for sirtuins and nicotinamide-producing enzymes. Anal Biochem 2009 Nov 01;394(1):101-9
Date
07/21/2009Pubmed ID
19615966Pubmed Central ID
PMC2752052DOI
10.1016/j.ab.2009.07.019Scopus ID
2-s2.0-69249206539 (requires institutional sign-in at Scopus site) 112 CitationsAbstract
Nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high-throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to alpha-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340 nm. Using this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of k(cat) and K(m) values with the native acetylated substrate acetyl-CoA synthetase-1; measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts; and determination of IC(50) values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide-forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.
Author List
Smith BC, Hallows WC, Denu JMAuthor
Brian C. Smith PhD Associate Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
ADP-ribosyl Cyclase 1Amino Acid Sequence
Animals
Cattle
Cell Extracts
Cell Line
Enzyme Inhibitors
Enzymes
Glutamate Dehydrogenase
Humans
Kinetics
Linear Models
NAD
NADP
Niacinamide
Peptides
Sequence Homology
Sirtuins
Spectrometry, Fluorescence
Spectrum Analysis
Time Factors